An evo-devo geek's scientific meanderings

ancestor reconstruction

Time to reexamine some assumptions (again)! And also, talk about Hox genes, because do I even need a reason?

Hox genes often come up when we look for explanations for various innovations in animal body plans – the digits of land vertebrates, the limbless abdomens of insects, the various feeding and walking and swimming appendages of crustaceans, the strongly differentiated vertebral columns of mammals, and so on.

Speaking of differentiated vertebral columns, here’s one group I’d always thought of as having pretty much the exact opposite of them: snakes. Vertebral columns are patterned, among other things, by Hox genes. Boundaries between different types of vertebrae such as cervical (neck) and thoracic (the ones bearing the ribcage) correspond to boundaries of Hox gene expression in the embryo – e.g. the thoracic region in mammals begins where HoxC6 starts being expressed.

In mammals like us, and also in archosaurs (dinosaurs/birds, crocodiles and extinct relatives thereof), these boundaries can be really obvious and sharply defined – here’s Wikipedia’s crocodile skeleton for an example:

In contrast, the spine of a snake (example from Wikipedia below) just looks like a very long ribcage with a wee tail:

Snakes, of course, are rather weird vertebrates, and weird things make us sciencey types dig for an explanation.

Since Hox genes appear to be responsible for the regionalisation of vertebral columns in mammals and archosaurs, it stands to reason that they’d also have something to do with the comparative lack of regionalisation (and the disappearance of limbs) seen in snakes and similar creatures. In a now classic paper, Cohn and Tickle (1999) observed that unlike in chicks, the Hox genes that normally define the neck and thoracic regions are kind of mashed together in embryonic pythons. Below is a simple schematic from the paper showing where three Hox genes are expressed along the body axis in these two animals. (Green is HoxB5, blue is C8, red is C6.)

As more studies examined snake embryos, others came up with different ideas about the patterning of serpentine spines. Woltering et al. (2009) had a more in-depth look at Hox gene expression in both snakes and caecilians (limbless amphibians) and saw that there are in fact regions ruled by different Hoxes in these animals, if a little fuzzier than you’d expect in a mammal or bird – but they don’t appear to translate to different anatomical regions. Here’s their summary of their findings, showing the anteriormost limit of the activity of various Hox genes in a corn snake compared to a mouse:

Such differences aside, both of the above studies operated on the assumption that the vertebral column of snakes is “deregionalised” – i.e. that it evolved by losing well-defined anatomical regions present in its ancestors. But is that actually correct? Did snakes evolve from more regionalised ancestors, and did they then lose this regionalisation?

Head and Polly (2015) argue that the assumption of deregionalisation is a bit stinky. First, that super-long ribcage of snakes does in fact divide into several regions, and these regions respect the usual boundaries of Hox expression. Second, ordinary lizard-shaped lizards (from which snakes descended back in the days of the dinosaurs) are no more regionalised than snakes.

The study is mostly a statistical analysis of the shapes of vertebrae. Using an approach called geometric morphometrics, it turned these shapes from dozens of squamate (snake and lizard) species into sets of coordinates, which could then be compared to see how much they vary along the spine and whether the variation is smooth and continuous or clustered into different regions. The authors evaluated hypotheses regarding the number of distinct regions to see which one(s) best explained the observed variation. They also compared the squamates to alligators (representing archosaurs).

The results were partly what you’d expect. First, alligators showed much more overall variation in vertebral shape than squamates. Note that that’s all squamates – leggy lizards are nearly (though not quite) as uniform as their snake-like relatives. However, in all squamates, the best-fitting model of regionalisation was still one with either three or four distinct regions in front of the hips/cloaca, and in the majority, it was four, the same number as the alligator had.

Moreover, there appeared to be no strong support for an evolutionarypattern to the number of regions – specifically, none of the scenarios in which the origin of snake-like body plans involved the loss of one or more regions were particularly favoured by the data. There was also no systematic variation in the relative lengths of various regions; the idea that snakes in general have ridiculously long thoraxes is not supported by this analysis.

In summary, snakes might show a little less variation in vertebral shape than their closest relatives, but they certainly didn’t descend from alligator-style sharply regionalised ancestors, and they do still have regionalised spines.

Hox gene expression is not known for most of the creatures for which vertebral shapes were analysed, but such data do exist for mammals (mice, here), alligators, and corn snakes. What is known about different domains of Hox gene activation in these three animals turns out to match the anatomical boundaries defined by the models pretty well. In the mouse and alligator, Hox expression boundaries are sharp, and the borders of regions fall within one vertebra of them.

In the snake, the genetic and morphological boundaries are both gradual, but the boundaries estimated by the best model are always within the fuzzy boundary region of an appropriate Hox gene expression domain. Overall, the relationship between Hox genes and regions of the spine is pretty consistent in all three species.

To finish off, the authors make the important point that once you start turning to the fossil record and examining extinct relatives of mammals, or archosaurs, or squamates, or beasties close to the common ancestor of all three groups (collectively known as amniotes), you tend to find something less obviously regionalised than living mammals or archosaurs – check out this little figure from Head and Polly (2015) to see what they’re talking about:

(Moving across the tree, Seymouria is an early relative of amniotes but not quite an amniote; Captorhinus is similarly related to archosaurs and squamates, Uromastyx is the spiny-tailed lizard, Lichanura is a boa, Thrinaxodon is a close relative of mammals from the Triassic, and Mus, of course, is everyone’s favourite rodent. Note how alligators and mice really stand out with their ribless lower backs and suchlike.)

Although they don’t show stats for extinct creatures, Head and Polly argue that mammals and archosaurs, not snakes, are the weird ones when it comes to vertebral regionalisation. For most of amniote evolution, the norm was the more subtle version seen in living squamates. It was only during the origin of mammals and archosaurs that boundaries were sharpened and differences between regions magnified. Nice bit of convergent/parallel evolution there!

ParaHox genes are a bit like the underappreciated sidekicks of Hox genes. Or little sisters, as the case may be, since the two families are closely related. Hox genes are probably as famous as anything in evo-devo. Being among the first genes controlling embryonic development to be (a) discovered, (b) found to be conserved between very distantly related animals, they are symbolic of the late 20th century evo-devo revolution.

ParaHoxes get much less attention despite sharing some of the most exciting properties of Hox genes. Like those, they are involved in anteroposterior patterning – that is, partitioning an embryo along its head to tail axis. Also like Hox genes, they are often neatly clustered in the genome, and when they are, they tend to be expressed in the same order (both in space and time) in which they sit in the cluster*. Their main ancestral roles for bilaterian animals seem to be in patterning the gut and the central nervous system (Garstang and Ferrier, 2013).

There are three known types of ParaHox gene, which are generally thought to be homologous to specific Hox subsets of Hox genes – by the most accepted scheme, Gsx is the closest sister of Hox1 and Hox2, Xlox is closest to Hox3, and Cdx to Hox9 and above. It is abundantly clear that Hoxes and ParaHoxes are closely related, but there has been a bit of debate concerning the number of genes in the ancestral gene cluster that gave rise to both – usually called “ProtoHox” (Garcia-Fernàndez, 2005).

Another big question about these genes is precisely when they originated, and in this regard, ParaHox genes are proving much more interesting than Hoxes. You see, there are plenty of animals with both Hox and ParaHox genes, which is what you’d expect given the ProtoHox hypothesis, but there are also animals with only ParaHoxes. If there really was a ProtoHox gene/cluster that then duplicated to give rise to Hoxes and ParaHoxes, then lone ParaHoxes (or Hoxes for that matter) shouldn’t happen – unless the other cluster was lost along the way.

So a suspiciously Gsx-like gene in the weird little blob-creature Trichoplax, which has nothing remotely resembling a Hox gene, was a big clue that (a) Hox/ParaHox genes might go back further in animal evolution than we thought, (b) the loss of the entire Hox or ParaHox cluster is totally possible**, despite how fundamental these genes appear to be for correctly building an animal.

I wrote (at length) about a study by Mendivil Ramos et al. (2012), which revealed that while Trichoplax had no Hox genes and only one of the three types of ParaHox gene, it preserved the more or less intact genomic neighbourhoods in which Hox and ParaHox clusters are normally situated. One of the more interesting results of that paper was that the one sponge genome available at the time – that of Amphimedon queenslandica, which had no trace of either Hox or ParaHox genes – also contained statistically significant groupings of Hox and ParaHox neighbour genes, as if it had a Hox neighbourhood and a ParaHox neighbourhood, but the Hoxes and ParaHoxes themselves had moved out.

That study thus pointed towards an intriguing hypothesis, previously championed by Peterson and Sperling (2007) based solely on gene phylogenies: sponges once did have Hox and ParaHox genes/clusters, which at least some of them later lost. This would essentially mean that the two gene clusters go straight back to the origin of animals if not further***, and we may never find any surviving remnant of the ancestral ProtoHox cluster, since the closest living relatives of animals have neither the genes nor their neighbourhoods (that we know of).

Hypotheses are nice, but as we know, they do have a tendency to be tragically slain by ugly facts. Can we further test this particular hypothesis about sponges? Are there facts that could say yay or nay? (Of course there are. I wouldn’t be writing this otherwise 😉 )

I keep saying that we should always be careful when generalising from one or a few model organisms, that we ignore diversity in the animal kingdom at our own peril, and that “distantly related to us” = “looks like our distant ancestors” is an extremely dodgy assumption. Well, here’s another lesson in that general vein: unlike Amphimedon, some sponges have not just the ghosts of vanished ParaHox clusters, but intact, honest to god ParaHox genes!

It’s calcareous sponges again. Sycon ciliatum and Leucosolenia complicata, two charming little calcisponges, recently had their genomes sequenced (alas, they weren’t yet public last time I checked), and since then, there’s been a steady stream of “cool stuff we found in calcisponge genomes” papers from Maja Adamska’s lab and their collaborators. I’ve discussed one of them (Robinson et al., 2013), in which the sponges revealed their rather unhelpful microRNAs, and back in October (when I was slowly self-destructing from thesis stress), another study announced a couple of delicious ParaHoxes (Fortunato et al., 2014).

(Exciting as it is, the paper starts by tickling my pet peeves right off the bat by calling sponges “strong candidates for being the earliest extant lineage(s) of animals”… I suppose nothing can be perfect… *sigh*)

The study actually covers more than just (Para)Hox genes; it looks at an entire gene class called Antennapedia (ANTP), which includes Hoxes and ParaHoxes plus a handful of related families I’m far less interested in. Sycon and Leucosolenia don’t have a lot of ANTP genes – only ten in the former and twelve in the latter, whereas a typical bilaterian like a fruit fly or a lancelet has several times that number – but from phylogenetic analyses, these appear to be a slightly different assortment of genes from those present in Amphimedon, the owner of the first sequenced sponge genome. This picture is most consistent with a scenario in which all of the ANTP genes in question were present in our common ancestor with sponges, and each sponge lineage lost some of them independently. (You may not realise this until you start delving into the history of various gene families, but genes come and go a LOT in evolution.)

Sadly, many of the branches on these gene trees are quite wonky, including the one linking a gene from each calcisponge to the ParaHox gene Cdx. However, somewhat fuzzy trees are not the only evidence the study presents. First, the putative sponge Cdxes possess a little motif in their protein sequences that is only present in a handful of gene families within the ANTP class. If you take only these families rather than everything ANTP and make trees with them, the two genes come out as Cdx in every single tree, and with more statistical support than the global ANTP trees gave them. Another motif they share with all Hoxes, ParaHoxes and a few of their closest relatives, but not with other ANTP class families.

Second, at least the gene in Sycon appears to have the right neighbours (Leucosolenia was not analysed for this). Since the Sycon genome sequence is currently in pieces much smaller than whole chromosomes, only four or so of the genes flanking ParaHox clusters in other animals are clearly linked to the putative Cdx in the sponge. However, when the researchers did the same sort of simulation Mendivil Ramos et al. (2012) did for Amphimedon, testing whether Hox neighbours and ParaHox neighbours found across all fragments of the genome are (a) close to other Hox/ParaHox neighbours or randomly scattered (b) mixed or segregated, they once again found cliques of genes with little overlap, indicating the once-existence of separate Hox and ParaHox clusters.

Fortunato et al. (2014) also examined the expression of their newfound Cdx gene, and found it no less intriguing than its sequence or location in the genome, although their description in the paper is very limited (no doubt because they’re trying to cram results on ten genes into a four-page Nature paper). The really interesting activity they mention and picture is in the inner cell mass of the young sponge in its post-larval stages – the bit that develops into the lining of its feeding chambers. Which, Adamska’s team contend, may well be homologous to our gut lining. In bilaterians, developing guts are one of the major domains of Cdx and ParaHox genes in general!

So at least three different lines of evidence – sequence, neighbours and expression – make this picture hang together quite prettily. It’s incredibly cool – the turning on their heads of long-held assumptions is definitely the most exciting part of science, I say! On the other hand, it’s also a little disheartening, because now that everyone in the animal kingdom except ctenophores has definitive ParaHox genes and at least the empty seats once occupied by Hox genes, are we ever going to find a ProtoHox thingy? May it be that it’ll turn up in one of the single-celled beasties people like Iñaki Ruiz-Trillo are sequencing? That would be cool and weird.

The coolest twist on this story, though, would be to discover traces of ProtoHoxes in a ctenophore, since solid evidence for ProtoHox-wielding ctenophores would (a) confirm the strange and frankly quite dubious-sounding idea that ctenophores, not sponges, are the animal lineage farthest removed from ourselves, (b) SHOW US A FREAKING PROTOHOX CLUSTER. (*bounces* >_> Umm, * cough* OK, maturity can suck it 😀 ) However, given how horribly scrambled at least one ctenophore genome is (Ryan et al., 2013), that’s probably a bit too much to ask…

***

Notes

*Weirdly, the order of expression in time is the opposite of that of the Hox cluster. In both clusters, the “anterior” gene(s), i.e. Hox1-2 or Gsx, are active nearest the front of the embryo, but while anterior Hox genes are also the earliest to turn on, in the ParaHox cluster the posterior gene (Cdx) wakes up first. /end random trivia

**Of course we’ve long known that losing a Hox cluster is not that big a deal, but previously, all confirmed losses occurred in animals with more than one Hox cluster to begin with – a fish has plenty of Hox genes left even after chucking an entire set of them.

(I think we’ve reached the point where it’s weird to say happy new year. I could swear xkcd had a pertinent chart of funny, but I couldn’t find it.)

Once upon a time, I briefly mentioned the problematic relationships of hemichordates. Since a short paper bearing on the subject came out relatively recently (i.e. in December, yes, I’m far behind the times ;)), I thought I’d revisit it.

To begin, let’s orient ourselves on my trusty old animal phylogeny.

Hemichordates are a phylum of deuterostomes, and their closest relatives appear to be echinoderms like starfish. The inside of Deuterostomia looks something like this:

Hemichordates come in two flavours: the butt-ugly (but nevertheless intriguing) acorn worm, which even the artistic eye of 19th century zoologists couldn’t make appealing (a selection of them from Johann Wilhelm Spengel’s work below):

… and the slightly nicer-looking pterobranch. Well. They’re kind of fluffy. That counts as “nicer,” right? (A couple of Cephalodiscus from the Halanych lab below):

Acorn worms and pterobranchs have different bodies adapted to very different lifestyles. Pterobranchs are stalked, tentacled filter-feeders that often clone themselves into colonies that live together in a branching tube system. Acorn worms are solitary burrowers without tentacles, tubes or shells. Hemichordates possess features in common with vertebrates, such as gill slits, and they seem a lot less freakish than their sister phylum Echinodermata. So hemichordates are kind of the natural go-to group to look for properties of the deuterostome common ancestor.

The only problem is, to do that, you need a solid understanding of hemichordate phylogeny itself. Because there are two very different kinds of hemichordates, you have to first figure out which of those best represents their common ancestor: the sit-at-home plankton sifter or the roaming mud-eating worm. (Maybe neither. Wouldn’t that be funny.) And, as it happens, there’s some disagreement about that.

One view, espoused by the mighty zoological tome of Brusca and Brusca (2002) among others, puts acorn worms and pterobranchs as separate sister groups, and considers pterobranchs the more conservative of the two. The Bruscas write, on page 869, that “the enteropneusts [= acorn worms] have lost [their tentacles], no doubt in connection with their development of an infaunal lifestyle.” In this view, the deuterostome ancestor was a sessile filter feeder, and the long worm-like body and general moving-aboutiness of other deuterostomes is a new feature.

The other hypothesis, backed by DNA sequence data (Cannon et al., 2009)* and more recently the discovery of a tube-dwelling acorn worm from the Cambrian (Caron et al., 2013), is that pterobranchs are a weird subgroup of acorn worms and therefore unlikely to say much about our own distant ancestors.

One thing that AFAIK both camps agree on is that the ancestral acorn worm had a larva that looked nothing like an acorn worm. That’s something pretty common for marine invertebrates. Creatures as different as sea urchins and ragworms explore the seas by way of tiny, planktonic larvae that later metamorphose into a completely different animal**. (Tornaria larva of an unidentified hemichordate below by Alvaro E Migotto from the Cifonauta image database.)

However, the specific family of acorn worms that pterobranchs supposedly come from does not have such a larval stage. They develop more or less directly from fertilised eggs into mini-acorn worms.

Pterobranchs are poorly studied, so not much is known about their babies. Are they like the conventional acorn worm larva, with its distinctive body plan and curly rows of cilia? Or are they more straightforward precursors of the adult, like their presumed closest cousins? Stach (2013) describes a larva of the pterobranch Cephalodiscus gracilis that looks more like the latter. He found the minuscule creature crawling around in a colony of adult Cephalodiscus, and used thin sections and transmission electron microscopy to make a 3D reconstruction of it.

(His account of finding the baby makes me wonder how the hell he knew it did belong to Cephalodiscus. If my experience with tube-dwelling marine invertebrates is anything to go by, being found in a certain animal’s home is no guarantee that you’re related to said animal. I suppose, incomplete though they may be, older descriptions of pterobranch babies were good enough to identify the little guy?)

The image that emerges is of a rather featureless little sausage. According to Stach, it has a through gut, one full-fledged and one partially formed gill opening (asymmetry like that is not unheard of in deuterostome embryos/larvae), as well as a body cavity and a bunch of muscle cells. What it doesn’t have is any trace of the bands of cilia that “typical” acorn worm larvae use to swim and feed, nor some other structures (e.g. nerve centres) that characterise such larvae.

Taken at face value, this would suggest (assuming this is a typical pterobranch larva) that the pterobranchs-are-acorn worms people are right. I have my reservations, and not just because a sample size of one makes me statistically nervous. Using this description as evidence for evolutionary relationships assumes that traditional larvae with ciliary bands are hard to lose. But that’s quite possibly not the case.

Echinoderm larvae, for example, have changed a lot even in the last few million years. The changes occurred many times independently, and often involved a return from a full-fledged larval stage to more direct development (Raff and Byrne, 2006). I don’t know whether acorn worms display a similar sort of flexibility. How many have even been studied in terms of development?

So: detailed internal structure of a pterobranch larva? Cool. As to the worms first hypothesis… “consistent with” would be a better description than “supports”, I think.

Damn. Mistaking evolution for a ladder with us on top is something I fully expect from people who don’t study it for a living, but when evolutionary scientists make that mistake, it drives me apeshit. And they do it all the fucking time.

I don’t think most of them are aware of it. You’ve got to be really watching for the trap to have a chance of avoiding it. I slip every now and then, and then I spot it and rage at myself and get deeply philosophical about human nature and such. It’s such an easy and convenient thing to do. (Think of evolution as a ladder, not get philosophical, I mean.) It’s the way we’ve been conditioned to think since the first time we heard about evolution.

For most of the history of biology, no one blinked twice if you talked with culturally sanctioned anthropocentrism about “lower animals” or “higher vertebrates”. Evolution was a highway of progress, and some creatures just got further along than others. Naturally, we were speeding along right at the front.

Nowadays, I think most biologists who have to consider evolution in their work would tell you that evolution doesn’t work like that. The papers I read rarely contain such explicit references to the “march of progress”. (Can I call it the MOP?) However, that doesn’t mean the references are gone. They’ve just become so subtle that, I suspect, not even the people who make them realise they’re there.

It’s “basal lineages”. “Phylogenetically more primitive” creatures. Or “early-branching organisms”. Or “evolutionary old animals”. All of these are real terms used in real papers published this year. They aren’t restricted to bad papers. And if you stop to think about it, none of them make any goddamned sense.

Let’s picture an evolutionary tree first. I can’t really use my usual tree with all its question marks, but the one below, which I nicked from Srivastava et al. (2008), will do:

The “base” of the tree is to the left, where animals, Monosiga and fungi have their last common ancestor. (That was a long time ago.) “Basal” means close to the base. The branching point (node) that separates animals from the non-animals at the top is the basalmost node in this tree. The node that separates the sponge from the other animals is also a pretty basal node. The creature that gave rise to both sponges and other animals was a truly basal animal.

Now, which is the basal lineage?

The correct answer is “relative to what?”

Every node divides the tree into two lineages. It doesn’t make any sense to say that one of them is more basal than the other. There’s a basal node in the tree of animals. Sponges are on one side of that, the rest of the animals are on the other. If you take a vertebrate species, sponges are the last animal lineage you’ll encounter if you trace its ancestry back towards the base of the tree. If you take a sponge species, the lineage with vertebrates (and lots of other things) on it will be the last.

“Basal lineage” depends on your point of view.

Maybe actually taking the sponge point of view will help illustrate this. This tree comes from a paper about sponges (Sperling et al., 2010):

Unlike the previous tree, its branches are labelled with larger groups rather than species, but these represent more or less the same range of creatures. Monosiga from tree one is a choanoflagellate. Amphimedon is a haplosclerid demosponge, on the second branch from the bottom. Every other animal from the first tree is compressed down into that one branch labelled “Eumetazoans”. (OK, Trichoplax is not a eumetazoan, but that’s a technicality that doesn’t affect the point.) From this angle, it’s rather harder to see sponges as a basal animal lineage!

Equally, sponges are just as old as non-sponge animals, so calling them “old” is a tad dodgy. Here, you could argue that sponges have been around longer than, say, vertebrates, which is true to the best of our knowledge. In that sense, “sponges” is an older lineage than “vertebrates”. But that only means that “sponges” should be compared to “non-sponges” rather than “vertebrates”, and anyone making such comparisons should be as aware of the diversity lurking within sponges as they are of the diversity of other animals.

The “evolutionary old animals” quote actually comes from a paper that looked at stem cell genes in Hydra to understand the evolution of stem cells in animals. (Hemmrich et al., 2012). It’s not comparing cnidarians (the phylum hydras belong to) to something genuinely younger than them. I can’t resist quoting the whole offending sentenc:

Our observations provided new and comprehensive insight into the complex network that orchestrates patterning and tissue homeostasis in an evolutionary old animal that branched off almost 600 million years ago. (p3277)

Honestly, what does that even mean? Branched off from what?

OK, I know it means from our own ancestors. But my point is that this should not be taken for granted, and if you do take a human-centric point of view, you should bloody well make that explicit. You should not write as though evolution had some sort of “main branch” leading to us from which things split every now and then. Lineages split from each other.

You might think that I’m being pedantic just to have an excuse to rant, but the implicit views underlying examples like the above have real consequences for the study of evolution. Namely, they might lead scientists to assume that representatives of “basal” lineages got stuck in the Precambrian and could just stand in for their distant ancestors. This is dangerous.

Take sponges. Yes, in many respects they probably resemble the first animals more than we do. Chances are those ancient animals didn’t have sophisticated organs and like two hundred different cell types. However, chances also are that they were made of distinct cells rather than huge merged syncytia, and that they didn’t have elaborate skeletons made of some sort of mineral, both of which are properties of many sponges. All animals alive today had exactly the same amount of time to evolve their own quirks since their last common ancestor. We shouldn’t just assume that anything “simple” in an animal we regard as “basal” is inherited straight from that ancestor just because it fits our favourite story.

Case in point: the Amphimedon genome was found to be impoverished in many families of developmentally important “master” genes, and this fit nicely into the prevailing view of the increasing complexity of animals throughout their history (Larroux et al., 2008). But it’s likely that at least some of those genes were actually lost by Amphimedon‘s ancestors and not gained by ours (Mendivil Ramos et al., 2012). Assuming that “basal” (relative to us) means “similar to ancestor X” can very easily lead to unwarranted conclusions, and that can hinder our ability to figure out what really happened. To me, that’s a big deal.

***

References:

Hemmrich G et al. (2012) Molecular signatures of the three stem cell lineages in Hydra and the emergence of stem cell function at the base of multicellularity. Molecular Biology and Evolution29:3267-3280

Nature has a way of screwing with the human desire for order and simplicity. One of the most egregious examples I’ve encountered is the contrast between what you learn about animal development and, well, reality.

Bilaterian animals have been traditionally divided into two great groups: protostomes and deuterostomes. If I whip out my trusty little animal phylogeny, deuterostomes are indicated right there in the middle, and protostomes are the sum of the ecdysozoans and lophotrochozoans. (Whoever came up with the latter name, urgh. I have to think twice before I say or type it, and I’ve been into them for years.)

The names “protostome” and “deuterostome” were nicked from ancient Greek and literally mean “first mouth” and “second mouth”. They refer to where the mouth comes from during the development of these animals. Animal embryos go through a process called gastrulation, during which the initially ball- or disc-like embryo folds in to form a pocket, the primitive gut or archenteron. The archenteron connects to the outside through a single hole, the blastopore. In protostomes, supposedly, the mouth comes straight from the blastopore, hence “first mouth”. In deuterostomes, the blastopore forms the anus while the mouth opens somewhere else. (Or that’s what they teach you at school anyway)

Here’s a really cool animated gif of gastrulation in sea urchins from Stanford University’s sea urchin embryology resource. Note how the mouth appears late in the process and joins up with the archenteron – sea urchins are good and proper deuterostomes! (The red dots are the cells that form the larval skeleton, in case you wondered)

And here are real sea urchin embryos before and during gastrulation (source):

(Sea urchins are neat.)

That sounds nice and simple and clear-cut, which should be a big warning sign that it’s too neat to be true. As far as I know, deuterostomes are relatively well-behaved in this respect, but protostomes… protostomes are horribly misnamed. Hejnol and Martindale (2009) compiled a table of what’s known about the various openings in different bilaterian phyla, and found protostomes to be all over the place. In protostome embryos, the blastopore may become the mouth, the anus, both or neither, and this often varies even within a phylum.

In Hejnol and Martindale’s book chapter, priapulids (a. k. a., and I’m not kidding, penis worms) are listed as “?blastopore closure”. Well, when Andreas Hejnol and friends looked more closely at priapulid embryos, that became “nope, definitely deuterostomy”. Not only does the priapulid blastopore become an anus, its surroundings also express genes associated with butthole formation, and the “mouth” genes are active on the opposite side of the embryo. (Below: Priapulus developmental stages, from Martin-Durán et al. [2012] The forming mouth [mo] and anus [an] are marked by shiny green concentrations of actin protein.)

That’s not just another addition to an already long list of deuterostomous protostomes, the study argues. Priapulids are considered to be one of the more conservative phyla among the ecdysozoans. With nematodes for comparison that’s not saying much, but this study suggests that they are quite conservative in terms of gut developmental genetics. The authors also note that deuterostomy was likely the ancestral condition for both nematodes + nematomorphs and arthropods + water bears + velvet worms (I’m not sure how strong that inference is based on what they write about the above groups). That means it’s likely to have been the way the last common ancestor of ecdysozoans developed. Given that deuterostomes are deuterostomous, the ancestral ecdysozoan probably was, and arrow worms, a weird ?protostome phylum that’s probably neither ecdysozoan nor lophotrochozoan, also are, this seems to suggest that all bilaterians came from a deuterostomous ancestor. (Below: the front end of an arrow worm, just for the heck of it. Yvan Perez, Wikimedia Commons.)

And that speaks against one of the more popular theories on how bilaterians came about from a jellyfish-like ancestor. Cnidarians such as jellyfish and coral polyps have only one gut opening, which is derived from the blastopore. A popular (and, IMO, quite appealing) idea is that this opening elongated in the ancestors of bilaterians, and separate mouths and anuses came from the long slit-like blastopore closing in the middle. If the last common ancestor of bilaterians is shown to be deuterostomous, this proposition remains without evidence.

There’s always a catch, though. Remember what I (or rather, Hejnol and Martindale) said about variation within phyla? Well, priapulids today are not the most diverse phylum to put it mildly, but there are still sixteen known species in two extant classes. Martin-Durán et al. (2012) examined one. You know the obvious question: how do the others develop?

It occurs to me that with so few living species, priapulids are among the few phyla for which this question could be answered completely 😀

Gods, this is what I’m faced with all the time. Someone needs to tell me how proper science bloggers pick articles to discuss, because I just get my RSS alerts, start squeeing, and end up not writing about anything because damn, I WANT TO WRITE ABOUT EVERYTHING!

I give up. I’ll just dump all the cool stuff that’s accumulated on my desktop and bookmark bar here and return to lengthy meandering whenever I don’t feel like I’ve been caught in a bloody tornado 😉

So, here is some Cool Stuff…

(1) A group measured the rate of DNA decay in 158 moa bones of known age from three sites. Really cool stuff, to go out and directly measure how ancient DNA disappears from dead things under more or less identical conditions. The unsurprising result is that DNA decays exponentially, a bit like radioactive material. This suggests that the main cause of the decay is random breaking of the strands. The surprising bit is that this happens much more slowly than previously estimated, suggesting that in ideal (read: frozen) conditions, it might be worth looking for preserved DNA in samples as old as a million years.

(On a side note, if you ever get a chance to see a talk by Eske Willerslev, one of the authors and a leading expert on ancient DNA, don’t miss it. The man is absolutely hilarious.)

(2) The beaks of the finches, ormixing and matching developmental recipes. This study examines the genetic basis of beak shape in three little birds closely related to Darwin’s famous finches. The three finches, just like Darwin’s, share the same basic beak shape, only bigger or smaller. However, there seem to be two distinct developmental programs at work, using different genes and parts of the skeleton to orchestrate beak development. One of the three newly investigated species (the one most closely related to Darwin’s finches) apparently uses the same developmental program as its more famous relatives, even though its beak is shaped more like the other two birds studied here. I told you – genetics, development and homology are complicated 😉

(3) Armoured fossil links worm-like molluscs to chitons. There’s a little-known group (or groups) of molluscs called aplacophorans that have only a coat of tiny spicules instead of shells and look more like worms than “proper” molluscs. Exactly where they fit into our picture of mollusc evolution has been controversial to say the least – they could represent an old lineage separate from other molluscs, they could be related to cephalopods, they could be related to chitons, they could be one group or they could be two lineages in completely different places on the tree… Well, a new fossil named Kulindroplax seems to argue for the chiton connection: the animal has the characteristic armour plates of a chiton on an aplacophoran-like body. Similar creatures have been discovered before, but this guy with its detailed 3D preservation provides the clearest evidence of the link so far.

(4) More cool fossils – this time straight from my beloved Cambrian. Nereocaris, a newly described Burgess Shale arthropod, suggests to its discoverers that the earliest arthropods weren’t predators prowling the seafloor, but swimmers who might have been filter feeders and certainly weren’t predators. The animal has a bivalved shell around its front end, similar to many other Cambrian swimming arthropods, and a long abdomen with paddles at the end. It bears the arthropod hallmark of a hardened and jointed exoskeleton, but it lacks specialised limbs such as antennae or mouthparts. In a cladistic analysis of arthropods and their nearest relatives, the new species comes out on the first branch within true arthropods, and the next few branches as we move towards living arthropods all contain similar shelled, swimming creatures. Since the non-arthropods closest to the real thing (i.e. anomalocaridids) were also fin-tailed swimmers, this arrangement makes the transition between them and true arthropods smoother than previously thought. It also suggests that the hard exoskeleton so characteristic of arthropods originally functioned in swimming – perhaps as an anchor for swimming muscles.

Gods, it’s been so hard to keep my mouth shut about this. A friend of mine just published a paper about Hox genes, and I’ve known about it for a while and it’s been keeping me crazy excited because it’s fascinating and, well: Hox genes! Now that it’s finally out, I can blather about it to my heart’s content, and so I will. Be prepared for a long ride 😉

First of all, a quick rundown of Hox genes for those who aren’t evo-devo geeks. These genes encode transcription factors – proteins that switch genes on/off. They are members of the large and distinguished class of homeobox genes, many of which play important roles in orchestrating embryonic development. Hox genes in particular are famous for laying out the plan for the head to tail axes of bilaterian animals, and for often sitting in neat clusters in the genome and being expressed along the body axis in the same order they are in the cluster. (Below: one of my favourite scientific figures ever, a fruit fly embryo stained in different colours for each of its Hox genes*. From Lemons and McGinnis [2006] via Pharyngula) In short, Hox genes are fucking awesome and extremely important to boot.

Tracing origins

One of the unresolved questions about Hox genes is exactly where they come from, and the new study draws some interesting conclusions regarding their origins. Before we delve into Mendivil Ramos et al. ( 2012) itself, perhaps it’s best to pull out my old sketch of animal phylogeny, because the relationships of the great old animal lineages are kind of important for the discussion. So this is the family tree of animals at first approximation (photos were all sourced from Wikimedia Commons; more info about them in my Nectocaris post):

Mendivil Ramos et al. follow one of the more popular resolutions of the question marks, in which cnidarians are closest to bilaterians and placozoans are the sister group to cnidarians+bilaterians. They don’t talk too much about ctenophores, but I’ll return to that later 🙂

Bilaterians all have Hox genes, and in most of them they do what they were originally discovered doing in fruit flies: patterning the anterior-posterior axis as they say in Jargonese. Some bilaterians have duplicated individual genes or even whole Hox clusters (we have four clusters, and salmon have as many as 13), but it’s pretty uncontroversial that a neat Hox cluster with representatives of most existing types of Hox genes was present already on the left side of the bilaterian box. So was the little sister of the Hox cluster, unimaginatively called the ParaHox cluster, which only contains three kinds of genes but operates in a similar way to its more famous sister (Brooke et al., 1998).

Where did Hox and ParaHox genes come from? Given the phylogeny of the genes, it’s likely that there was originally a small (maybe 2-3 genes) ProtoHox cluster that duplicated to give rise to both Hoxes and ParaHoxes. We know that cnidarians like sea anemones have both Hox and ParaHox genes, which behave somewhat like their bilaterian counterparts (Ryan et al., 2007). Therefore, the ProtoHox cluster must have existed before the common ancestor of these two great lineages.

Enter the Blob

What about placozoans? That’s where things get a bit complicated. Trichoplax, the mysterious little blob that is the only living representative of this oddball phylum, has only one Hox-like gene noncommittally named Trox-2. A relic of the ProtoHox era? Not really – in phylogenetic analyses of the protein sequence, it tends to group with the ParaHox gene Gsx, whereas you would expect a leftover ProtoHox gene to remain outside the Hox+ProtoHox clique.

Is Trox-2 a ProtoHox gene anyway? That would mean something weird happened in the evolution of Hox and ParaHox genes after the cluster duplication: Gsx (and its sisters Hox1-2) would have stagnated somewhere near its ancestral condition while all the other genes sped ahead. It’s a long shot, but evolution has been known to do strange things to gene sequences. Also, homeobox genes are often difficult to classify by sequence alone. Scientists typically use the DNA-binding region that the homeobox encodes for this purpose, but a homeodomain is only 60 amino acids and simply doesn’t contain enough information to place some problematic sequences. And unless we’re examining very closely related genes, the rest of the protein sequence is too different to be compared.

Guilt by association

However, there is another way of solving the mystery. Hox and ParaHox genes are not alone in the genome. They sit on huge chromosomes, and while they tend to banish non-*Hox genes from among them, the flanks of each cluster are populated by a variety of unrelated genes. The key thing is that Hox clusters and ParaHox clusters have different neighbours. Thus, looking at a problem gene’s neighbours can tell us what it is!

(Above: the neighbours of Trox-2. Yellow genes are ParaHox neighbours in humans, green genes are Hox neighbours, grey genes have no human counterparts, and orange genes are parts of both Hox and ParaHox neighbourhoods. From Mendivil Ramos et al. [2012])

This is exactly what happened. My lovely friend Olivia looked at the chunk of genomic sequence that contains Trox-2 and found about two dozen genes on it that had clear homologues in humans. She then tallied where each of the human homologues were, and behold: many of them crowded around ParaHox clusters (we also have several of those, courtesy of whole genome duplications), while only one was a Hox neighbour in humans. If Trox-2 were a ProtoHox, we’d expect a mixture of Hox and ParaHox neighbours, but that’s not what we find at all. Statistically speaking, it’s a no-brainer. Trox-2 is exactly where a ParaHox gene should be.

Ghosts in the genome

Now, we have a problem. If Trox-2 is a ParaHox gene, it must have come after the Hox/ParaHox duplication. So where the hell is the Hox cluster? Well, seeing as Trichoplax only has one ParaHox gene instead of the more typical three or so, gene loss certainly sounds like a possibility. Is there an “empty” Hox cluster lurking somewhere in the blob’s genome? Here, cnidarians turn out to be pretty helpful. After sequencing the genome of the sea anemone Nematostella vectensis, Putnam et al. (2007) attempted to reconstruct parts of the original chromosomes of the cnidarian-bilaterian ancestor. They called the results Putative Ancestral Linkage Groups, in other words, groups of genes that have stayed together since cnidarians and bilaterians diverged 600 or so million years ago.

One of these PALs contains over 200 conserved Hox neighbours, nearly all of which are present in Trichoplax. Strikingly, about half of them are close enough to one another that they are in the same chunk of sequence even though the Trichoplax genome hasn’t been stitched together to the level of whole chromosomes. That’s much more than you’d expect by chance. Trichoplax has a Hox locus without Hox genes, what Mendivil Ramos et al. call a ghost Hox locus.

Hox genes all the way down?

If you followed so far, you might have noticed that we’ve been pushing that elusive ProtoHox further and further back in animal evolution. It preceded bilaterians, it preceded cnidarians and bilaterians, and now it turns out it also preceded our split from placozoans. Will we find it if we look in the remaining animal lineages? Since a ctenophore genome hasn’t yet been released to the public, that question transforms into: will we find it in sponges?

The sponge Amphimedon queenslandica does have a publicly available genome, and much has been made of its apparent lack of many developmentally important transcription factor families (e.g. Larroux et al., 2008). It doesn’t have anything that looks like a Hox, ParaHox or ProtoHox gene, but what about the neighbourhoods?

Like that of Trichoplax, the Amphimedon genome sequence is in relatively small pieces, so a little clever statisticking was needed to decide whether it contains Hox, ParaHox or ProtoHox neighbourhoods. The starting points were the PAL of Hox neighbours mentioned above, and a PAL of ParaHox neighbours the team constructed using the human and Trichoplax genomes. These genes were distributed among many genomic scaffolds, but of course lacking chromosome-level information the group didn’t know whether any of these scaffolds are actually linked to each other in the sponge genome.

The solution was a simulation: take the number of genes in the PAL, take the number and size (in number of genes) of the thousands of Amphimedon scaffolds, and scatter the PAL members randomly among the scaffolds with the larger scaffolds proportionately more likely to receive a PAL gene. When all the PAL members are handed out, count the number of scaffolds with PAL members on them. Repeat this a thousand times, and you get an idea what the distribution of Hox and ParaHox neighbours would be if they weren’t clustered together. This approach showed that the real distribution is anything but random. Hox and ParaHox neighbours are clearly clustered in the sponge genome, and what’s more, they are clustered separately.

Still no ProtoHox locus, in other words. At some point in the murky depths of their ancestry, sponges lost bona fide Hox and ParaHox genes!

So…

That raises a couple of issues. First, where is the ProtoHox? Hox-like genes have never been found outside animals. These are smart people we’re talking about, so they checked the genome of the closest non-animal relative we have today, a choanoflagellate. Neither Hox/ParaHox nor ProtoHox neighbourhoods were there – the PAL genes didn’t cluster together any more than they would by chance. The whole *Hox phenomenon seems unique to animals (or else the choanoflagellate genome is totally scrambled). It appears that somewhere in our ancestry, ProtoHox gene(s) appeared and parted ways before sponges split from the rest of the animals. Since we have no surviving descendants of these ancestors outside of sponges and the rest of the animals, we’ll probably never find unduplicated descendants of the ProtoHox cluster.

Second, what happened in ctenophores? Everything we know about their genomes suggests that they completely lack Hox-like genes. Although there have been studies that placed them even further out than sponges (Dunn et al., 2008), it’s more likely that they are much closer to bilaterians than that (Philippe et al., 2011). I think I’m not the only one itching to examine a ctenophore genome for Hox neighbours…

And finally, if some distant ancestor of all animals had full-blown Hox and ParaHox clusters, what the heck was it doing with them? Was it something unexpectedly complex that would need genes for axial patterning? Are sponges and placozoans grossly simplified descendants of a much more complex ancestor, or did Hox-like genes only become involved in dividing up body axes later in evolution?

The more we learn the less we know. One thing is (once again) clear: assuming that a simple animal is a good proxy for an ancestral animal is a dangerous, dangerous assumption to make.

***

*Technically, fruit flies have twelve Hox genes, but only seven are shown in the image. Hox2/proboscipedia is a normal Hox gene involved in the development of mouthparts among others, but four more genes have completely lost their “canonical” Hox gene-like activities. That includes all three of Drosophila‘s weird triplicated Hox3 genes.

I bumped into Mayer et al. (2010) while hunting for reagents to use in an experiment I’m planning. The article is about segmentation (sort of), so I had to have a closer look, and man. Those pictures. Fluorescence and a good microscope can do wonders. This is what a velvet worm looks like in normal light (whole body shot of an unspecified peripatid by Geoff Gallice, Wikipedia, and portrait of Euperipatoides rowelli by András Keszei via EOL):

I think they are adorable and cuddly the way they are (apart from that hunting with slime bit), but they look simply gorgeous if you stick some glowing antibodies to them and start playing with a confocal ‘scope.

This is a fairly late-stage embryo of E. rowelli, the same guy waving its chubby leggies at you in the right-hand photo above. The green dots are cells that were copying their DNA when the baby was killed (all of the pictures below are from Mayer et al., of course):

These are younger embryos of the same species, with all their DNA labelled in blue and dividing cells labelled in red:

And these are embryos of another species from the same family as the unidentified guy from Wikipedia (colours are the same as above):

Seriously, there is something about the mystical glow of these images that always gets me. I think you could make almost anything look beautiful with a fluorescent marker and the right equipment. I know aesthetic appeal isn’t the primary aim of scientific imaging, but damn. Look at those alien creatures glowing with the light of the unknown.

In case you wondered, the point of the paper is that velvet worms lack a posterior growth zone. That means that when they develop their numerous segments, there isn’t a well-defined pool of cells at the rear of the embryo that divide to generate segment material. As you can see in all the red glow, cell division happens evenly all over the place. Why is this significant? Well, posterior growth zones were thought to be one of the characteristics that segmented animals might have inherited from their common ancestor. But Mayer and colleagues point out that the existence of a PGZ in the arthropod ancestor is dubious at best, and velvet worms (one of the closest living relatives of arthropods) also lack one, so maybe it’s kind of wrong to use the PGZ as an argument for the common ancestry of segmentation.

(There, that’s the science in a nutshell. Now I’ll just go back and admire the pretty glowy pictures some more :D)
***

If you’ve ever visited this blog before, you probably know that the early evolution of animals is one of my many random interests. You could say it’s my main interest, though that may be less obvious from my posting record so far. Well, knowing that, you could imagine my face when my labmate pointed me to this National Geographic news piece.

It doesn’t surprise me much that the earliest known animal would be like a sponge. Although for what they do, their construction is nothing short of ingenious, sponges are comparatively simple animals. While it’s possible that they weren’t always like that, it appears that their genomes are devoid of lots of the genes other animals have added to the “toolkit” that fashions their complex bodies (Larroux et al., 2008). They also retain morphological features that were probably present in the ancestors of animals and lost in pretty much every other animal lineage alive today. Notably, their food-capturing cells look an awful lot like the cells of choanoflagellates, which are thought to be the closest living relatives of animals and perhaps similar in appearance to our distant ancestors.

What looks positively amazing about the newly described sponge-like thingies, who go by the deceptively Italian-sounding name of Otavia (they’re actually named after the Otavi Group of rock formations in Namibia), is their age. The oldest ones, apparently, are close to 760 million years old, perhaps 180 million years older than the earliest occurrences of the famous and mysterious Ediacaran animals (Narbonne, 2005). (By the way, that difference is about the length of the “age of dinosaurs”!) The news, and Brain et al. (2012), point out that this date also precedes some events that were thought to set the stage for the rise of animals: the giant ice ages known as Snowball Earths, and the rise in atmospheric oxygen levels towards the end of Precambrian times.

We could talk about the significance of that, I suppose, but the issue the whole discovery brought to my mind is, strangely, molecular clocks.

Let’s face it, the Precambrian fossil record of animals is not brilliant. It’s getting better, as more Ediacaran fossils are dug up and analysed with more sophisticated methods, but it still raises as many questions as it answers, and the earliest history of animals is still shrouded in mystery. For one thing, when did animals even evolve? All we know from fossils is that it must have been “before”. If a particular fossil is not only an animal, but member of an identifiable subgroup of animals, it means that the branch separating that subgroup from all other animal lineages must have split by that time. A number of Precambrian animals may be members of groups that are many such splits into the animal family tree, and things that look like the predecessors of those splits are difficult to identify in the fossil record. So where did they come from? Where did it all begin? Kind of hard to say based on the bunch of hard to interpret blobs, fronds and strange fractal bodies that is the Ediacaran biota.

When the fossil record speaks gibberish, people sometimes query another keeper of deep evolutionary history: DNA. Molecular clock methods date splits between lineages by counting differences between their living members. The basic idea is this: if most mutations have no effect on fitness, then most mutations are created equal, with the same chance of fixing themselves in the gene pool. If that is true, then genomes change at roughly constant rates – dependent only on mutation rate – over time. Using that assumption and lineages whose divergence time is known (usually, from good fossil records), you can translate the genetic differences between two or more groups into evolutionary time.

The problem is that real life is not so simple as that. Evolution is not always neutral. The same gene may behave like a clock in one lineage or during one time period and not another. Part of a gene may be a good clock while another part isn’t. Even if all of a gene evolves in a clock-like manner in all lineages under study, there’s no guarantee that the clock will tick at the same rate in all of them. Different genes or parts of a gene can tick at different rates, and this can vary over time. If we’re trying to measure very long times, it can be hard to correctly estimate the amount of change in a gene. There can be error in the fossils used for calibration, or the calibrating lineages may evolve differently from the ones we’re interested in. And so on.

And thus, published estimates for early divergences among animals range from numbers that make reasonable sense with the fossil record (e.g. Peterson et al., 2004), to some that throw another billion years on top of those numbers (see Chapter 11 in Knoll [2003] for an accessible discussion).

The problem, as I see it, is this. With a billion-year margin of error, some of those estimates must be wrong. As Andrew Knoll noted, they all require that animals began much earlier than their fossil record (at least as it was known at the time). How can we trust any of them? Even for the ones that match what we think of the fossil record – well, stopped clocks are accurate twice a day. For a scientist, being accidentally right is no better than being wrong.

I suppose Otavia, if it’s really a sponge-ish creature, fits the Peterson & co. estimates quite neatly. Fairly certain bilaterians like Kimberella are known from the White Sea assemblage of the Ediacaran, somewhat under 560 million years ago (Narbonne, 2005). The origin of bilaterians is somewhere between two and four splits[1] after sponges diverged from other animals. Peterson and colleagues estimated it between 573 and 656 million years ago – so if sponges are indeed a conservative bunch, sponge-like animals must have been around quite a bit earlier, but perhaps >1 billion years ago is really stretching it. 760 million sounds kind of nice, farther back than the Kimberellas and Dickinsonias but not too far.

Kind of. But, seeing as we’ve had to wait this long for a maybe-sponge that old, who’s to say even older animals aren’t hiding in some unexplored fossil bed? Who’s to say that the next “oldest animal” find won’t validate some of the more outlandish estimates?

The other thing I’m wondering about re: Otavia is: is it a sponge (assuming it’s an animal at all), or could it belong to a lineage ancestral to both sponges and other animals? (Were early sponges ancestral to other animals? The idea has been played with in phylogenetic circles…) I guess we’ll never know for certain. I still think it’s worth raising the question. Creatures that might be ancestral to more than one phylum are extremely valuable to evolutionary biologists, but they might be very hard to recognise for what they are. Part of the problem with Ediacaran animals is that many if not most of them lack features associated with living phyla – but that’s exactly what we would expect from creatures that preceded the divergence of those phyla! Given how little, say, a jellyfish and a snail have in common, what on earth would their common ancestors look like? Would they have any fossilisable characteristics at all that could give us a hint as to their family ties?

And I guess I’ll close today’s musings with that question. If I spent more time reading Brain et al. (2012), there’d probably be a lot more to discuss, but after doing lab work all day and spending an extra couple of hours writing this, my brain doesn’t feel up to it 🙂

***

[1] You can refer to the rough animal phylogeny in the Nectocaris post for the moment. Being slightly out of the loop in this area, I wouldn’t hazard a guess as to the relationships of ctenophores, cnidarians and placozoans, hence my uncertainty. It’s possible that these three all form a single branch with bilaterians on the other side. Or they could represent three different branching events, or anything in between. I really should make an animal phylogeny page, I think, since I keep finding myself wanting to talk about bilaterians and lophotrochozoans and things that don’t make much sense unless you know at least the basics shown in tree I made for Nectocaris…

Behold the lengthy going-on about limbs, developmental genetics and semi-philosophical stuff that I promised! I mentioned that this was long in the making. Ironically, that means I’m not sure I managed to make it coherent… Then again, my blog subtitle does warn you about certain “meanderings” 😉

***

I previously mentioned that limbs kind of brought me to evo-devo. I haven’t closely followed the subject since, but a recent paper (Schneider et al., 2011) brought it back to my attention. Aside from the nostalgia, the evolution of limbs is also a perfect excuse for me to ruminate on some of the issues I consider important in evo-devo – such as the meaning of evidence, the role of “model organisms” and the nature of homology and novelty. (Some of this I touched on in my treehopper post)

I love developmental genetics. Davis et al. (2007), which through the blurred glasses of hindsight I’ll call the paper that made me an evo-devo nerd, is a genetic study. Genes are really exciting for us evolutionists because they obey different rules from the traits they control. Especially for regulatory genes – those that affect the activity of other genes –, gene sequence doesn’t correspond to the appearance of the organism in any straightforward way. The same circuitry of regulatory genes can also control the development of quite different structures, because most of the actual work is done by their target genes. Therefore, genes can often preserve connections we can no longer see in higher-level traits. (My favourite combination of evidence is genes plus fossils, but bear with me a little…)

The gist of Davis et al. (2007) is as follows. Hands and feet (collectively known as the autopod) are unique to tetrapods, or vertebrates with legs. There’s a special pattern of Hox gene activity that controls autopod formation. This pattern was missing from the fish that had been examined at the time. However, those fish are quite different from the distant ancestors they shared with tetrapods. There are living fish whose fin skeletons include bits that might correspond to digits, and there are many fossil examples. These include, as it later turned out, not just the iconic fishapod Tiktaalik, but also its slightly less tetrapod-like relative Panderichthys (Boisvert et al., 2008). Hence the question: did common lab animals like zebrafish lose the bones and the genetic circuitry, and did the bones of the autopod evolve from particular bones of the ancestral fin, or did tetrapods invent something new?

The answer is almost certainly the former, Davis et al. (2007) tell us after they find the tetrapod kind of Hox gene expression in the fins of a comparatively “primitive” ray-finned fish (Ray-fins are one of the two main groups of bony fishes. The zebrafish is a ray-fin, as are other familiar fish like cod and tuna. The other group – lobe-fins – include lungfish, coelacanths and tetrapods themselves). Around the same time, other teams found similar patterns in lungfish (Johanson et al., 2007), which are probably the closest living relatives of tetrapods, and sharks (Freitas et al., 2007), which are only distantly related to any of the creatures mentioned above.

Schneider et al. (2011), which caused this post, found that some DNA elements that regulate the Hox genes in the autopod are shared by tetrapods, ray-fins and sharks (ergo, probably all living vertebrates with fins or limbs). Together with the evidence from fossil and modern skeletons, this suggests that the digits of tetrapods evolved from pre-existing fin bones by tweaking an ancient genetic program. Fins and limbs really are variations on a single ancient theme. (Illustration of “fishapod” fins and early tetrapod limbs below is by Dennis C Murphy, from Devonian Times)

It is at this conclusion that we come to the stuff Hox gene expression can’t tell us. Knowing that radial bones (or cartilages, as the case may be) in fins and digits in limbs are “really the same thing” in some way is one thing. But radials and digits are not that similar, and neither is a shark’s fin and a newt’s leg. Maybe you’re interested in how one became the other, how fins suited to balancing and manoeuvring in water became limbs suited to plodding along on land. The autopod-like Hox pattern doesn’t say, since it’s basically the same in appendages that look very different, a perfect example of what I said about regulatory genes a few paragraphs back. Clearly, Hox genes define a distinct part of the fin or limb, but they don’t give detailed instructions on how to flesh it out. The details depend on the genes under Hox control.

Unseen ancestors

Now, fins and limbs are relatively easy, because we have a really quite awesome fossil record of their history (and also, some cool computer models :D). But the same lessons we can learn from their example apply equally to countless other cases where the fossil record is silent. Shared expression patterns of “master” genes and genetic pathways are often used to infer things about ancestors that aren’t known from fossils at all (De Robertis, 2008 is a nice review of such pathways). How far can we take such inferences? What does the fact that arthropods, vertebrates and segmented worms all seem to use some of the same genetic pathways to generate their bodies from repeating units (e.g. Stollewerk et al., 2003; Pueyo et al., 2008; Rivera and Weisblat, 2009)? Was their common ancestor as obviously segmented as an earthworm, did it just have a few repeated body parts like a chiton, or maybe nothing more than the basic ladder-like nervous system* of bilaterian animals? Or perhaps even less?

[*Photo of the nervous system of a planarian flatworm stained with a fluorescent dye, by the Agata group.]

The fossil record of early animal evolution (or rather, the lack of it) argues that this common ancestor was relatively small and simple (Erwin and Davidson, 2002). We know that quite different structures can be underpinned by the same “master” genes. Given this, can we really say anything meaningful about such long-extinct creatures? Well, we certainly can. They probably had the genetic circuitry their descendants share today. But what does that say about their body plans?

The answer may not be too far from “fuck all”. That’s why I chose a quote from Tabin et al. (1999) for the title of this post. I couldn’t agree more when they write, “developmental genetics only tells us what characters might have been possible”. I love finding out where we and the other creatures with whom we share this planet came from. That’s why I’m in this business. But there is only so much that any given type of evidence can tell us. And this is why I think the fossil record is so important. Like Erwin and Davidson (2002) argue, it can help us distinguish between “might have beens” in sometimes surprising ways.

Same difference

All of this puts the whole concept of homology into a slightly unsettling new perspective. Homologues (often spelled “homologs” nowadays) are supposed to be traits (genes, organs, behaviours etc.) that are derived from the same ancestral trait. The original concept of homology was defined for whole organs/body parts. Now, what do we do with organs that are made by the same genetic networks? Some of them show obvious historical continuity with the organs of other organisms. A bird’s wing is clearly homologous to my arm, on probably every level imaginable. They are connected by similar position on the body, similar basic structure, similar development and developmental genetics, and a rich fossil record. But that absolutely need not be the case.

Some butterflies use the same genetic circuitry to put eyespots on their wings that insects in general use to subdivide their wings into different regions (Keys et al., 1999). It would be quite absurd to call wings and eyespots homologous because of that – but in a very real sense, the gene network underpinning both is “the same thing”. And there is everything in between. Eyes, for example, share common “master” developmental genes including Pax6/eyeless. They were probably built around homologous cell types (i.e. photoreceptors) in most animals that have them (e.g. Arendt, 2003). Nonetheless, the highly complex eye structures of, say, a squid, a dragonfly and a falcon almost certainly evolved independently. And then there are the strange, confused identities of bird fingers that I talked about on previous occasions. Thus, when we ask the question: “are these two structures homologous?” – there is often no simple yes/no answer. At the very least, you have to ask: at what level?

An ode to diversity

The whole autopod business also highlights the dangers of extrapolation. Scientists believed that the autopod-specific Hox code was invented by tetrapods because their staple experimental fish didn’t have it. But life is a huge, diverse bush. Every twig has its own unique quirks, and we can’t take any of them to represent everything on its branch in every respect. In fact, some of the most popular lab animals – fruit flies, nematode worms, the aforementioned zebrafish – are also among the quirkier denizens of the planet. This is why I find it really really important not to limit ourselves to a few well-worn “model organisms”, not to draw sweeping conclusions from them. Although our common ancestry means that fruit flies or nematodes will in many ways help us understand ourselves, there is no guarantee. Comparative biology thrives on diversity.

(Of course, I say that as an evolutionary biologist working on a non-model organism. I may be somewhat biased ;))

***

References

Arendt D (2003) Evolution of eyes and photoreceptor cell types. The International Journal of Developmental Biology47:563-571

Boisvert CA et al. (2008) The pectoral fin of Panderichthys and the origin of digits. Nature456:636-638

Davis MC et al. (2007) An autopodial-like pattern of Hox expression in the fins of a basal actinopterygian fish. Nature447:473-476